Identifying new antibiotics is critical for the long-term control of multi-drug resistant tuberculosis (TB). Several new classes of compounds that kill Mycobacterium tuberculosis (Mtb) have been discovered in recent years, including inhibitors of the MmpL3 mycolic acid transporter. MmpL3 is a common target of small molecule inhibitors of mycobacterial growth identified by whole cell, phenotypic screens. It is a mycolic acid flippase that moves trehalose monomycolate (TMM) to the pseudoperiplasmic space, from where TMM is further modified to trehalose dimycolate (TDM) and incorporated into the mycomembrane. Mtb and M. smegmatis mmpL3 knockdown strains provide genetic evidence that mmpL3 is essential for survival both in vitro and in a mouse TB infection model. This essentiality makes MmpL3 an attractive therapeutic target and supports efforts to characterize small molecules targeting MmpL3. Multiple MmpL3 inhibitors also exhibit synergistic interactions with TB antibiotics, further supporting interest in this target. Using an innovative combination of untargeted and targeted mutant screens, we have identified ten new and distinct scaffolds that function by inhibiting MmpL3 function. These compounds are bactericidal both in vitro and against intracellular Mtb in primary murine macrophages. Mutations in mmpL3 provided resistance to these compounds. Mtb cells treated with these compounds were shown to accumulate TMM and have reduced levels of TDM, further supporting their targeting MmpL3 activity. Cluster analysis of cross-resistance profiles, defined two clades of inhibitors and two clades of resistant mutants. Pairwise combination studies of the inhibitors revealed antagonistic, synergistic and additive interactions that were specific to the identified clades. The combined study of multiple mutants and new compounds provides new insights into structure-function interactions of MmpL3 and its inhibitors and will enable the development of new classes of Mtb inhibitors. The goal of this proposal is to conduct exploratory studies to develop new, more durable compounds that inhibit MmpL3. Compounds will be optimized for potency and pharmacokinetic (PK) properties and tested for efficacy in an acute mouse model of Mtb infection (Aim 1). Additionally, we will conduct studies examining the frequency of resistance of Mtb treated with combinations of MmpL3 inhibitors (Aim 2). We hypothesize that specific combinations of inhibitors, based on differences in their interactions with MmpL3, may reduce the frequency of resistance or select for resistant mutants with reduced fitness. This finding would support the development a more durable, hybrid MmpL3 inhibitor sharing the combined activity of the paired treatments. New chemical matter and proof-of-concept data generated in these studies will derisk further development of a new MmpL3 inhibitor that can treat MDR-TB. OVERALL IMPACT: This project will optimize novel Mtb inhibitors, define their function in vivo and identify combinations of compounds that will reduce frequency of drug resistance.